Low Pressure Gel Permeation Chromatography Research Articles

Chromatographic Analytical Control and Purification of Water-Soluble Carboxylated Copolymers Based on N-Vinylamides

Chromatographic methods of analysis of the products of the radical copolymerization of random copolymers of N-vinyl-2-pyrrolidone and 6-crotonoyl aminohexanoic acid are developed and molecular characteristics of the copolymers are determined. A method is proposed for the purification of the obtained carboxylated water-soluble copolymers of monomeric hydrophobic acid impurities by low-pressure gel-permeation chromatography.

Preparation of Hybrid Fucoidan-derived Oligosaccharides and Their Structural Analysis by Negative-ion Electrospray Tandem Mass Spectrometry

Acomplex sulfated fucoidan(SPF) was obtained by hot water extraction from Sargassum pallidum.A series of hybrid oligosaccharides were acquired by mild acid hydrolysis with low pressure gel permeation chromatography(LPGPC).Based on the monosaccharides composition,methylation and electrospray collision-induced dissociation mass spectrometry(ES-CID-MS/MS) analysis,21 kinds of oligosaccharides were sulfated hybrid fucoidan-derived oligosaccharides.The resulted oligosaccharides were mainly composed of α1→3 linked Fuc,β1→4 linked Xyl and β1→6 linked Gal.The sulfation occurred at C4 or C2 of fucose,C2 of xylose and C4 of galactose residue,respectively.Most of fucose residues were present in nonreducing terminal of oligosaccharides.The technique of ES-CID-MS/MS was suitable for the sequences analysis of other hybrid oligosaccharides of various fucoidans.More importantly,all these diverse sulfated oligosaccharides can be printed onto the glycol-chip,and studying their interactions with proteins.

Hyperbranched polylysines: Mechanism of formation

To gain insight into the mechanism treating formation of hyperbranched polylysines through the polymerization of N ɛ-carbobenzoxylysine N-carboxyanhydride under conditions of the reductive removal of a N ɛ-carbobenzoxy group, hyperbranched polylysine has been synthesized with the use of trifluoroacetic acid as a terminator in the polymerization of N-carboxyanhydride. The structure of the polymers is studied by capillary electrophoresis, low-pressure gel-permeation chromatography, circular dichroism, and enzymatic hydrolysis with trypsin. At the first stage of synthesis, a low-molecular-mass strongly branched core of the polymer is formed. At the second stage, polylysine chains are grafted via one point onto amino groups of N-terminal lysine moieties of the low-molecular-mass core through their carboxyl ends.

The influence of molecular mass of sulfated propylene glycol ester of low-molecular-weight alginate on anticoagulant activities

Abstract Sulfated propylene glycol ester of low-molecular-weight alginate (PSS) was fractionated by low-pressure gel permeation chromatography. Four fractions (PSS1–4) with different weight-averaged molecular mass (51.95, 25.62, 11.76 and 5.41 kD, respectively) were obtained and their structural characteristics were determined and compared by Fourier transform infrared and nuclear magnetic resonance spectroscopes. Their anticoagulant activities were also studied by evaluating their influence on the activated partial thromboplastin time, thrombin time, anti-thrombin and anti-FXa factor activities mediated by anti-thrombin III and heparin cofactor II. The results demonstrated that PSS1–4 had similar structural parameters, such as the degree and pattern of sulfation and backbone, but their molecular masses were different. The bioassay results suggested that the anticoagulation was molecular mass dependent. PSS1 and PSS2 strongly inhibited the activity of thrombin mediated by heparin cofactor II and anti-thrombin III, whereas PSS4 only showed a very weak anti-thrombin activity. PSS1–3 weakly inhibited the activity of coagulation factor Xa mediated only by anti-thrombin III while PSS4 lacked the anti-Xa activity.

Preparation and structure elucidation of alginate oligosaccharides degraded by alginate lyase from Vibro sp. 510

Alginate that was purified from the fermentation solution of marine bacteria Vibro sp. 510 under specific reaction conditions was hydrolyzed by alginate lyase. Seven oligosaccharides, including di-, tri- and tetrasaccharides, were isolated through low-pressure, gel-permeation chromatography (LP-GPC) and semipreparative strong-anion exchange (SAX) fast-protein liquid chromatography (FPLC). The oligosaccharide structures were elucidated based on ESIMS and 2D NMR spectral analysis. The hydrolytic specificity of this alginate lyase to alginate is discussed.

Synthesis and characterization of novel water soluble amphotericin B–arabinogalactan conjugates

The coupling of amphotericin B (AmB), a water-insoluble antifungal agent, to arabinogalactan (AG) via an imine or amine bond was systematically investigated. AG was oxidized using potassium periodate, purified from the oxidizing agent using ion-exchange chromatography, and reacted with AmB to form the Schiff base. The Schiff base was reduced to the amine using borohydride. All reactions took place in aqueous media. The purification of the oxidized AG from the oxidizing agent was essential to prevent oxidative degradation of AmB at the coupling step. We investigated the effects of AmB to AG ratio, buffer type, and reaction pH on the reaction yield, molecular weight, conjugate activity against pathogenic yeast and hemolytic activity. The optimum coupling conditions were buffer borate 0.1 m, pH 11 at room temperature for 48 h. Lower toxicity in vivo was achieved by using low-pressure gel permeation chromatography and applying the solution of AmB–AG conjugate through a Sephadex column. Both amine and imine AmB–AG conjugates were soluble in water and exhibited improved stability in aqueous solutions as compared to the unbound drug. The conjugates showed comparable minimum inhibitory concentration (MIC) values against Candida albicans. The conjugates were about 60 times less hemolytic against sheep erythrocytes than the free drug, and about 40 times less toxic in BALB/c mice.

Characterization of coal-derived humic substances with the aid of low-pressure gel permeation chromatography

Low-pressure gel permeation chromatography (GPC) has proven to be a valuable tool for determining the molecular weights of coal-derived humic substances (HS). A 40×0.9 cm (ID) column packed with Sephadex gel (typically with Sephadex G-100) in combination with alkaline borate-based mobile phase allows an almost complete fractionation of typical HS from young brown coals. After a calibration with a set of globular proteins, a molecular-weight distribution can be determined and average molecular weights can be calculated. The determination of the weight-average molecular weight, M w, seems to be more reliable than the determination of the number-average molecular weight, M n. Limitations of the proposed method (and size-exclusion methods in general) were discussed in detail, and some sources of uncertainties were identified.

Preparation and structural determination of dermatan sulfate-derived oligosaccharides.

Eight oligosaccharides were prepared from dermatan sulfate (DS) and their structures were elucidated. Porcine intestinal mucosal DS was subjected to controlled depolymerization using chondroitin ABC lyase (chondroitinase ABC). The oligosaccharide mixture formed was fractionated by low-pressure gel permeation chromatography (GPC). Size uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, and dodecasaccharides were obtained. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange (SAX) high-performance liquid chromatography (HPLC). This approach has led to the isolation of six homogeneous oligosaccharides. The size of the oligosaccharides were determined using GPC-HPLC. Treatment of tetrasaccharide and hexasaccharide fragments with Hg(OAc)2 afforded trisaccharide and pentasaccharide products, respectively. The purity of the oligosaccharides obtained was confirmed by analytical SAX-HPLC, and capillary electrophoresis (CE). The molecular mass and degree of sulfation of the eight purified oligosaccharides were elucidated using electrospray ionization (ESI) mass spectrometry and their structures were established with high field nuclear magnetic resonance (NMR) spectroscopy. These DS-oligosaccharides are currently being used to study for interaction of the DS with biologically important proteins.

Determination of polyaromatic hydrocarbons and some related compounds in industrial waste oils by GPC-HPLC-UV

The determination of polyaromatic hydrocarbons (PAHs) in industrial waste oils was studied. The optimized procedure consisted in using low pressure gel permeation chromatography to separate the fraction containing PAHs and PCBs from the matrix. Both groups of contaminants were further fractionated on a small column of 3% deactivated alumina, using hexane to elute the PCB fraction and then hexane–dichloromethane (95 + 5) to elute PAHs. The final analysis was carried out by HPLC on a 2(1-pyrenyl)ethyldimethylsilica column with hexane as mobile phase in the isocratic mode and with UV detection at three different wavelengths, to quantify 16 priority PAHs. Recoveries were between 89 and 99%. Four different industrial waste oil samples were analyzed and the results obtained are discussed.

Soil organic matter mobilization by root exudates of three maize hybrids

In order to study the macromolecular behaviour of humic substances, we treated humic material (from the Ah horizon of an Eutric Cambisol) either with exudates from 10 day-old seedlings obtained in axenic conditions from three maize cultivars (Zea mays L.; cv. Paolo, cv. Polaris and cv. Sandek) or with the organic acids (succinic and fumaric) present in the exudates and followed the change in molecular size distribution. Succinic acid, Polaris and Sandek exudates shifted the humic absorbance from high to low molecular weight in size exclusion chromatograms, while Paolo exudates were not able to produce the same shift and gave an absorbance profile like that of humic substance alone. Our results are evidence of the micellar behaviour of humic substances in solution and of the importance of hydrophobic bonds in holding humic molecular together. Exudates were also used to separate soil organic fractions from the same soil. Soil organic extracts were characterized by low pressure gel permeation chromatography, hormone-like activities, and 1H-NMR. The data, compared with water extract and KOH extract, indicate that maize exudates, fumaric and succinic acids mobilize soil organic fractions in small quantities, but these extracts are endowed with auxin- and/or gibberellin-like activity. Water extract and maize exudates exhibit no hormone-like activity, while KOH extract shows a negligible one. Moreover, only Polaris and Sandek exudates and succinic acid are able to disaggregate an organic fraction with low apparent molecular size (<13KDs) from the bulk of the soil. The 1H-NMR data indicate that water and succinic extracts are largely composed of carbohydrate and aliphatic constituents. Sandek exudate and fumaric acid can liberate aromatic components, Polaris exudate releases carbohydrates from soil, while Paolo slightly influences the aliphatic constituents. The role of organic acids released by maize seedlings in mobilising soil organic fractions is presented.

Preparation and structure of heparin lyase-derived heparan sulfate oligosaccharides.

Porcine intestinal mucosal heparan sulfate was exhaustively depolymerized on a large scale using heparin lyase II (heparinase II) or heparin lyase III (heparitinase, EC 4.2.2.8). The oligosaccharide mixtures formed with each enzyme were fractionated by low pressure gel permeation chromatography. Size-uniform mixtures of disaccharides, tetrasaccharides, and hexasaccharides were obtained. Each size-fractionated mixture was then purified on the basis of charge by repetitive semipreparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 13 homogenous oligosaccharides. The purity of each oligosaccharide was demonstrated by the presence of a single peak on analytical strong-anion-exchange high-performance liquid chromatography and reversed polarity capillary electrophoresis. The structures of these oligosaccharides were established using 500 MHz one- and two-dimensional nuclear magnetic resonance spectroscopy. Three of the thirteen structures that were solved were novel while the remaining 10 have been previously described. All of the structures obtained using heparin lyase III contained a delta UAp residue (where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid) at their nonreducing termini. Structures obtained using heparin lyase II contained both delta UAp and delta UAp2S (where S is sulfate) at their nonreducing termini. These results are consistent with the reported specificity of both enzymes.

Macromolecular changes of humic substances induced by interaction with organic acids

SummaryTwo different humic materials, one from a forest soil and the other from wormcasts, were used to study the influence of mineral and organic acids on the conformational properties of humic substances. The macromolecular changes were followed by low pressure gel permeation chromatography after titrating humic material to low pHs with acids. All organic acids (mono‐, di‐, tri‐carboxylic, and oxy‐acids), added to humic solution prior to a gel permeation in an alkaline buffer, were able to shift the totality of absorbance of the humic chromatographic peak from high to low molecular sizes. Mineral acids, phenol, alcohols, dipolar aprotic solvents, could not produce the same shift and gave total absorbance at the column void volume as in the case of humic substances alone. The chromatographic peak shifted back to elution volumes proper of higher size molecules when the humic‐organic acid mixture was back‐titrated to high pHs before gel permeation. Elution in a much stronger alkaline buffer did not change the overall macromolecular behaviour.These results suggest that humic substances behave as micelles in solution and that hydrophobic bondings play an important role in holding humic molecules together. The organic acids enter the interior of the humic micelle‐like aggregates and alter the stereochemical hydrophobic arrangement of the humic material. In alkaline conditions the negative charges developed disrupt the apparent high molecular size configuration and disperse the humic aggregates into small micelles. Such conformational properties of humic substances appear to be a function of pH and of the concentration of organic acid.

Preparation and structural characterization of large heparin-derived oligosaccharides.

Porcine mucosal heparin was partially depolymerized with heparin lyase I and then fractionated into low-molecular-weight (< 5000) and high-molecular-weight (> 5000) oligosaccharides by pressure filtration. The high-molecular-weight oligosaccharide mixture (approximately 50 wt% of the starting heparin) also contained intact heparin. This intact polymer complicates oligosaccharide purification. Thus, the low-molecular-weight fraction was used to prepare homogeneous oligosaccharides for structural characterization. The low-molecular-weight oligosaccharide mixture was first fractionated by low-pressure gel permeation chromatography into size-uniform mixtures of disaccharides, tetrasaccharides, hexasaccharides, octasaccharides, decasaccharides, dodecasaccharides, tetradecasaccharides and higher oligosaccharides. Each size-fractionated mixture was then purified on the basis of charge by repetitive semi-preparative strong-anion-exchange high-performance liquid chromatography. This approach has led to the isolation of 14 homogeneous oligosaccharides from disaccharide to tetradecasaccharide. The purity of these heparin-derived oligosaccharides was determined by gradient polyacrylamide gel electrophoresis, analytical strong-anion-exchange high-performance liquid chromatography, capillary electrophoresis and one-dimensional nuclear resonance spectroscopy. The structure of these oligosaccharides was established using 600 MHz two-dimensional nuclear resonance spectroscopy. The spectral methods used included homonuclear correlation spectroscopy, nuclear Overhauser effect spectroscopy and heteronuclear multiple quantum coherence spectroscopy. The 1H/1H connectivities of the protons of each sugar residue in an oligosaccharide were established by two-dimensional homonuclear correlation spectroscopy, while 1H/13C assignments were made using 1H inverse detection. One- and two-dimensional nuclear resonance spectroscopic analysis of these heparin oligosaccharides showed two closely related groups of heparin-oligosaccharides are afforded by enzymatic depolymerization of heparin. One group is fully sulphated, having the structures delta UAp2S(1[-->4)-alpha-D-GlcNpS6S(1-->4)-alpha-L-IdoAp2S( 1]n-->4)-alpha- D-GlcNpS6S, where delta UAp is 4-deoxy-alpha-L-threo-hex-4-eno-pyranosyluronic acid, GlcNp is 2-deoxy-2-aminoglucopyranose, IdoAp is idopyranosyluronic acid, S is sulphate and n = 0-6. The other group of oligosaccharides differ in that they contain beta-D-glucuronic acid in place of the alpha-L-iduronic acid residue nearest to the reducing end. The present study describes the isolation and structural elucidation of seven new oligosaccharides: an octasaccharide, two decasaccharides, two dodecasaccharides and two tetradecasaccharides. The utility of two-dimensional nuclear resonance spectroscopy to determine the structure of complex heparin oligosaccharides is also illustrated.

High-performance liquid chromatographic separation of heparin-derived oligosaccharides

Heparin has been enzymatically depolymerized with heparinase (heparin lyase (EC 4.2.2.7)) and then separated into di-, tetra-, hexa-, octa-, and decasaccharide mixtures by low-pressure gelpermeation chromatography (GPC). These sized mixtures were resolved by strong anion-exchange (SAX) HPLC into multiple components. The fractions from the SAX-HPLC were collected and characterized for size by GPC-HPLC and sulfate content by ion chromatography. This study provides detailed methodology for the separation of larger and more highly sulfated oligosaccharides than previously reported. It describes the first use of ion chromatography for the accurate determination of the sulfate content of heparin oligosaccharides, a method which can also be applied to heparin and other glycosaminoglycans.